Ethanol is the most common substance of abuse in the US and an important risk-factor for infections such as bacterial pneumonias which are a leading cause of mortality in alcoholics. We, and others, have accumulated a body of evidence that acute ethanol induces suppression of macrophage tumor necrosis factor-alpha (TNF) secretion, which leads to immunosuppression. Chronic ethanol however, has been associated with an augmentation of stimulated TNF responses, which is associated with liver injury. Preliminary data in macrophage cell lines as well as Kupffer cells has identified that chronic EtOH trough a redox dependent epigenetic mechanism augments TNF transcription in response to EtOH. In part this is due to increase nuclear translocation of NF-kB. However preliminary studies using chromatin immunoprecipitation assays also show increased binding of the AP-1 family member FosB and interferon regulatory-3 to the TNF promoter and this binding is critical for augmented TNF transcription. Moreover, preliminary data suggest that chronic ethanol also results in defective TGF-beta repression of TNF-alpha transcription in part due to defective Smad3/4 nuclear translocation in the setting of chronic ethanol. Based on these data, we hypothesize that chronic ethanol exposure results in increased TNF transcription in macrophages by altering the cellular redox state which results in increased binding of both AP-1 and IRF-3 to the TNF promoter as well as reducing the nuclear level of Smad 3 and 4 which function as a TNF represser. We will test this hypothesis with the following Specific Aims. 1.1. Our hypothesis predicts that chronic ethanol results in a redox-dependent increase in AP-1 and IRF-3 binding to the human TNF promoter resulting in increased transcription of the TNF gene. 2. Investigate the hypothesis that loss of repression by Smad 3 and 4 contributes to increased TNF production by chronic ethanol. 3. Investigate the contributions of AP-1, Trif/IRF-3, and Smad 3 and 4 in regulating increased mononuclear cell TNF production in a mouse model of chronic ethanol. By understanding the complex regulation of ethanol on TNF transcription, we hope to advance the understanding of ethanol's effect on the immune system and enhance potential therapeutic strategies for ethanol related diseases.